Crystal Structure of a Yeast Aquaporin at 1.15 ? Reveals a Novel Gating Mechanism

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.     

A 2.1-Å-Resolution Crystal Structure of Unliganded CRM1 Reveals the Mechanism of Autoinhibition

CRM1 mediates nuclear export of numerous proteins and ribonucleoproteins containing a leucine-rich nuclear export signal (NES). Binding of RanGTP to CRM1 in the nucleus stabilizes cargo association with CRM1, and vice versa, but the mechanism underlying the positive cooperativity in RanGTP and NES binding to CRM1 remains incompletely understood. Herein we report a 2.1-Å-resolution crystal structure of unliganded Saccharomyces cerevisiae CRM1 (Xpo1p) that demonstrates that an internal loop of CRM1 (referred to as HEAT9 loop) is primarily responsible for maintaining the NES-binding cleft in a closed conformation, rendering CRM1 incapable of NES binding in the absence of RanGTP. The structure also shows that the C-terminal tail of CRM1 stabilizes the autoinhibitory conformation of the HEAT9 loop and thereby reinforces autoinhibition. Comparison with the structures of CRM1–NES–RanGTP complexes reveals how binding of RanGTP is associated with a series of allosteric conformational changes in CRM1 that lead to opening of the NES-binding cleft, allowing for stable binding of NES cargoes.     

The Crystal Structure of the Catalytic Domain of the NF-κB Inducing Kinase Reveals a Narrow but Flexible Active Site

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Highlights? Production of soluble NIK kinase domain ? Structures of apo murine and human NIK possess active conformation ? Structure of mNIK bound to inhibitors reveals conformational flexibility ? Inhibitor potency varies against mNIK and hNIK due to substitution in the active site     

Crystal Structure of Adenylylsulfate Reductase from Desulfovibrio gigas Suggests a Potential Self-Regulation Mechanism Involving the C Terminus of the β-Subunit

Adenylylsulfate reductase (adenosine 5′-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-Å resolution. Different from the α₂β₂-heterotetramer of the Archaeoglobus fulgidus, the overall structure of APSR from D. gigas comprises six αβ-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the α-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the α-helix (amino acid 289 to 299) in the α-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the β-subunit wraps around the α-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the α-subunit from another αβ-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the α-subunit and Asp159 in the C terminus of the β-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the β-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the β-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the β-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the β-subunit.Sulfate-reducing bacteria (SRB) are a special group of prokaryotes that are found in sulfate-rich environments because of their ability to metabolize sulfate. SRB use sulfate as the final electron acceptor in various anaerobic environments, such as soil, oil fields, the sea, or the innards of animals or even human beings (10, 11, 19, 25, 33). Their ability to degrade sulfate offers protection against environmental pollution. SRB can remove sulfate and toxic heavy atoms from factory waste waters (12). The Desulfovibrio species is a much-studied representative of SRB, and Desulfovibrio gigas has been studied under many diverse conditions to elucidate metabolic pathways (23, 35).Sulfate reduction is one of the oldest forms of cellular metabolism. The reduction can be either assimilatory or dissimilatory. Sulfate is the terminal electron acceptor in dissimilatory reduction and the raw material for the biosynthesis of cysteine in assimilatory reduction. The latter type of reduction occurs in archaebacteria, bacteria, fungi, and plants via various pathways (17). For example, in Escherichia coli, the reduction initially catalyzes sulfate to adenosine 5′-phosphosulfate (APS) by ATP sulfurylase. APS is then phosphorylated by APS kinase to 3′-phosphate APS, which is then further reduced to sulfite by 3′-phosphate APS reductase (APSR). Finally, sulfite is reduced by sulfite reductase to sulfide, which condenses with O-acetylserine by O-acetylserine lyase to form cysteine. For comparison, in dissimilatory sulfate reduction, sulfate is first catalyzed by ATP sulfurylase to APS, which is then directly reduced by APSR to sulfite. Sulfite is subsequently reduced by dissimilatory sulfite reductase to the following three possible products: trithionite (S₃O₆²⁻), thiosulfate (S₂O₃²⁻), or sulfide (S²⁻).Adenylylsulfate reductase, also called APSR, plays an important role in catalyzing APS to AMP and sulfite in the dissimilatory sulfate reduction. APSR was first partially purified and characterized from Desulfovibrio desulfuricans (32). Multiple forms of APSR in Desulfovibrio vulgaris were observed in buffers under varied conditions (1) and were found in the cytoplasm of cells (18). APSR from D. gigas was first purified by Lampreia et al. (21) and showed a molecular mass of 400 kDa comprised of α- and β-subunits, corresponding to the molecular masses of 70 kDa and 23 kDa, respectively. One flavin adenine dinucleotide (FAD) and two [4Fe-4S] clusters per APSR have been observed and characterized by electron paramagnetic resonance and Mössbauer spectroscopy. The enzyme from D. gigas has been described as an α₂β complex involving one FAD and two [4Fe-4S] clusters (20). In D. vulgaris, APSR is apparently an α₂β₂ complex with a molecular mass of 186 kDa; only one Fe-S cluster is found in the αβ-heterodimer (31). Thus, the subunit and quaternary structures of APSR and their constitution of cofactors in terms of FAD and iron-sulfur clusters are still under debate. Only the enzyme from Archaeoglobus fulgidus has benefited from having an X-ray crystal structure. In this APSR, the functional unit has been shown to be the 1:1 αβ-heterodimer, containing two iron-sulfur clusters and one FAD in the structure (7). However, crystal packing shows that the asymmetric unit is an α₂β₂-heterotetramer.The catalytic mechanism of APSR can be divided into the transport of electrons and the cleavage of APS by FAD. Electron input to the FAD catalyzes the cleavage of APS, releasing AMP and sulfite. Although there have been a number of mechanisms proposed to explain the catalytic cleavage of APS to AMP and sulfite (7, 8, 13, 20, 34), many features of the postulated mechanism remain unsettled, including the proteinogenic hydrogen acceptor in the reaction, the conformational change in the enzyme induced by reduction/oxidation of the FAD cofactor, and the reasons for the observed multiple forms of APSR. The divergence between A. fulgidus and Desulfovibrio species also suggests an obvious distinction in the phylogeny of the α- and β-subunits of APSR.To clarify the difference between APSR from A. fulgidus and that from Desulfovibrio species, we have undertaken a structural study of APSR from D. gigas for comparison with the A. fulgidus enzyme. We have isolated and purified APSR directly from massive, anaerobically grown D. gigas cells for structure determination and characterization. The comparison of the structures and sequences revealing the notable differences at the C termini, activity sites, and other regions for the function is discussed. The structure of oxidized APSR from D. gigas provides much direct evidence about the subunit interactions and the role of the quaternary structure in the regulation of the catalytic mechanism.     

High Resolution Crystal Structure of Human β-Glucuronidase Reveals Structural Basis of Lysosome Targeting

Human β-glucuronidase (GUS) cleaves β-D-glucuronic acid residues from the non-reducing termini of glycosaminoglycan and its deficiency leads to mucopolysaccharidosis type VII (MPSVII). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases. The structure revealed several new details including a new glycan chain at Asn272, in addition to that previously observed at Asn173, and coordination of the glycan chain at Asn173 with Lys197 of the lysosomal targeting motif which is essential for phosphotransferase recognition. Analysis of the high resolution structure not only provided new insights into the structural basis for lysosomal targeting but showed significant differences between human GUS, which is medically important in its own right, and E. coli GUS, which can be selectively inhibited in the human gut to prevent prodrug activation and is also widely used as a reporter gene by plant biologists. Despite these differences, both human and E. coli GUS share a high structure homology in all three domains with most of the glycosyl hydrolases, suggesting that they all evolved from a common ancestral gene.     

The Crystal Structure of the Pseudomonas dacunhae Aspartate-β-Decarboxylase Dodecamer Reveals an Unknown Oligomeric Assembly for a Pyridoxal-5′-Phosphate-Dependent Enzyme

The Pseudomonas dacunhael-aspartate-β-decarboxylase (ABDC, aspartate 4-decarboxylase, aspartate 4-carboxylyase, E.C. 4.1.1.12) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-decarboxylation of l-aspartate to produce l-alanine and CO₂. This catalytically versatile enzyme is known to form functional dodecamers at its optimal pH and is thought to work in conjunction with an l-Asp/l-Ala antiporter to establish a proton gradient across the membrane that can be used for ATP biosynthesis. We have solved the atomic structure of ABDC to 2.35 Å resolution using single-wavelength anomalous dispersion phasing. The structure reveals that ABDC oligomerizes as a homododecamer in an unknown mode among PLP-dependent enzymes and has highest structural homology with members of the PLP-dependent aspartate aminotransferase subfamily. The structure shows that the ABDC active site is very similar to that of aspartate aminotransferase. However, an additional arginine side chain (Arg37) was observed flanking the re-side of the PLP ring in the ABDC active site. The mutagenesis results show that although Arg37 is not required for activity, it appears to be involved in the ABDC catalytic cycle.     

The Crystal Structure of an Integral Membrane Fatty Acid α-Hydroxylase

Neuronal electrical impulse propagation is facilitated by the myelin sheath, a compact membrane surrounding the axon. The myelin sheath is highly enriched in galactosylceramide (GalCer) and its sulfated derivative sulfatide. Over 50% of GalCer and sulfatide in myelin is hydroxylated by the integral membrane enzyme fatty acid 2-hydroxylase (FA2H). GalCer hydroxylation contributes to the compact nature of the myelin membrane, and mutations in FA2H result in debilitating leukodystrophies and spastic paraparesis. We report here the 2.6 Å crystal structure of sphingolipid α-hydroxylase (Scs7p), a yeast homolog of FA2H. The Scs7p core is composed of a helical catalytic cap domain that sits atop four transmembrane helices that anchor the enzyme in the endoplasmic reticulum. The structure contains two zinc atoms coordinated by the side chains of 10 highly conserved histidines within a dimetal center located near the plane of the cytosolic membrane. We used a yeast genetic approach to confirm the important role of the dimetal-binding histidines in catalysis and identified Tyr-322 and Asp-323 as critical determinants involved in the hydroxylase reaction. Examination of the Scs7p structure, coupled with molecular dynamics simulations, allowed for the generation of a model of ceramide binding to Scs7p. Comparison of the Scs7p structure and substrate-binding model to the structure of steroyl-CoA desaturase revealed significant differences in the architecture of the catalytic cap domain and location of the dimetal centers with respect to the membrane. These observations provide insight into the different mechanisms of substrate binding and recognition of substrates by the hydroxylase and desaturase enzymes.     

Crystal Structure of α-1,4-Glucan Lyase,a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-d-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (β/α)₈-barrel catalytic domain with B and B′ subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp⁵⁵³ and Asp⁶⁶⁵ are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite −1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.     

Crystal Structure of the GalNAc/Gal-Specific Agglutinin from the Phytopathogenic Ascomycete Sclerotinia sclerotiorum Reveals Novel Adaptation of a β-Trefoil Domain

A lectin from the phytopathogenic ascomycete Sclerotinia sclerotiorum that shares only weak sequence similarity with characterized fungal lectins has recently been identified. S. sclerotiorum agglutinin (SSA) is a homodimeric protein consisting of two identical subunits of ∼ 17 kDa and displays specificity primarily towards Gal/GalNAc. Glycan array screening indicates that SSA readily interacts with Gal/GalNAc-bearing glycan chains. The crystal structures of SSA in the ligand-free form and in complex with the Gal-β1,3-GalNAc (T-antigen) disaccharide have been determined at 1.6 and 1.97 Å resolution, respectively. SSA adopts a β-trefoil domain as previously identified for other carbohydrate-binding proteins of the ricin B-like lectin superfamily and accommodates terminal non-reducing galactosyl and N-acetylgalactosaminyl glycans. Unlike other structurally related lectins, SSA contains a single carbohydrate-binding site at site α. SSA reveals a novel dimeric assembly markedly dissimilar to those described earlier for ricin-type lectins. The present structure exemplifies the adaptability of the β-trefoil domain in the evolution of fungal lectins.     

The Structure of the Cyprinid herpesvirus 3 ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L,a Poxvirus Inhibitor of Interferon Response

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.     

Recognition of the Helical Structure of β-1,4-Galactan by a New Family of Carbohydrate-binding Modules

The microbial enzymes that depolymerize plant cell wall polysaccharides, ultimately promoting energy liberation and carbon recycling, are typically complex in their modularity and often contain carbohydrate-binding modules (CBMs). Here, through analysis of an unknown module from a Thermotoga maritima endo-β-1,4-galactanase, we identify a new family of CBMs that are most frequently found appended to proteins with β-1,4-galactanase activity. Polysaccharide microarray screening, immunofluorescence microscopy, and biochemical analysis of the isolated module demonstrate the specificity of the module, here called TmCBM61, for β-1,4-linked galactose-containing ligands, making it the founding member of family CBM61. The ultra-high resolution x-ray crystal structures of TmCBM61 (0.95 and 1.4 Å resolution) in complex with β-1,4-galactotriose reveal the molecular basis of the specificity of the CBM for β-1,4-galactan. Analysis of these structures provides insight into the recognition of an unexpected helical galactan conformation through a mode of binding that resembles the recognition of starch.     

Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation

Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIδ/Ca²⁺/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIδ/Ca²⁺/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix αD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules.

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Atomic Structure of the Francisella T6SS Central Spike Reveals a Unique α-Helical Lid and a Putative Cargo

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Crystal Structure of TNF-α-Inducing Protein from Helicobacter Pylori in Active Form Reveals the Intrinsic Molecular Flexibility for Unique DNA-Binding

Tipα (TNF-α-inducing protein) from Helicobacter pylori is a carcinogenic effector. Studies on this protein revealed that a homodimer linked by a pair of intermolecular disulfide bridges (Cys25-Cys25 and Cys27-Cys27) was absolutely necessary for its biological functions. The activities of Tipα would be abolished when both disulfide bridges were disrupted. The crystal structures of Tipα reported to date, however, were based on inactive, monomeric mutants with their N-terminal, including residues Cys25 and Cys27, truncated. Here we report the crystal structure of H. pylori Tipα protein, TipαN²⁵, at 2.2Å resolution, in which Cys25 and Cys27 form a pair of inter-chain disulfide bridges linking an active dimer. The disulfide bridges exhibit structural flexibility in the present structure. A series of structure-based mutagenesis, biochemical assays and molecular dynamic simulations on DNA-Tipα interactions reveal that Tipα utilizes the dimeric interface as the DNA-binding site and that residues His60, Arg77 and Arg81 located at the interface are crucial for DNA binding. Tipα could bind to one ssDNA, two ssDNA or one dsDNA in experiments, respectively, in the native or mutant states. The unique DNA-binding activities of Tipα indicate that the intrinsic flexible nature of disulfide bridges could endow certain elasticity to the Tipα dimer for its unique bioactivities. The results shed light on the possible structural mechanism for the functional performances of Tipα.     

The Characterization of the Endoglucanase Cel12A from Gloeophyllum trabeum Reveals an Enzyme Highly Active on β-Glucan

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50°C on β-glucan. Under these conditions specific activity was 239.2±9.1 U mg⁻¹ and the half-life of the enzyme was 84.6±3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the K_m was 3.2±0.5 mg mL⁻¹ and the V_max was 0.41±0.02 µmol min⁻¹. Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.     

Characterization of ELP-fused ω-Transaminase and Its Application for the Biosynthesis of β-Amino Acid

Optically pure amines, β-amino acids and γ-amino acids are the valuable precursors to produce biologically active compounds. The ω-TAs are the class of enzymes which are widely used to produce such compounds. In this work (S)-ω-transaminase from the thermophilic eubacterium Sphaerobacter thermophilus (St-TA) was fused with Elastin-like polypeptides (ELPs) through the cloning process and expressed in E. coli cells. The characterization of this fusion complex was performed with respect to thermostability and effect of DMSO. Where in case of St-TA-ELP-V₆₀, major difference in the transition temperature (T_t) was observed, wherein a T_t of 38 and 70°C was observed at the increasing concentration of DMSO from 5 to 25% (v/v). Interestingly, these fusion proteins the activity was preserved even after the aggregation of fusion complex at Tt. The substrate specificity and product inhibition analysis showed that ω-TA-ELPs had comparable results as that of wild type ω-TA. Moreover, the fused ω-TA could be efficiently reused for up to 20 batches of transamination reaction. Furthermore, the applicability of the fusion protein for the production of a sitagliptin precursor (R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid (3-ATfBA) was evaluated, wherein 3-ATfBA was synthesized with good conversion (65%).